Peptide-chelate conjugates

ABSTRACT

A peptide-chelate with affinity for the ST receptor is disclosed, wherein the chelate is tetradentate. The peptide-chelate conjugate of the invention may be labelled with a radiometal to provide a metal complex. A radiopharmaccutical composition comprising the metal complex is provided, which is suitable for the diagnostic imaging of colorectal cancer. Also provided for in the invention is a kit for the preparation of the radiopharmaceutical preparation.

FIELD OF THE INVENTION

This invention relates to radiopharmaceuticals, in particular theinvention relates to a peptide-chelate conjugate, comprising a peptidewith affinity for the ST receptor. The compound of the invention issuitable for diagnostic imaging of colorectal cancer in a mammal.

The invention additionally relates to a radiolabelled peptide-chelateconjugate with affinity for the ST receptor, wherein radiolabelling ofthe peptide-chelate conjugate does not interfere with the affinity ofthe peptide for the ST receptor. In another aspect of the invention, useof such a compound for imaging of cancer of colorectal origin isprovided.

A kit for the production of a radiolabelled peptide-chelate conjugatefor imaging colorectal cancer is also disclosed.

BACKGROUND ART

Colorectal carcinoma (CRC) is the fourth most common malignancyworldwide following cancers of the lung, breast and prostate. Metastasesof CRC origin are the main cause of death in patients diagnosed withcolorectal primary tumours. Approximately 150,000 to 200,000 new casesare diagnosed annually in the USA and around 50,000 deaths areattributed to this disease. Many patients die due to the metastaticspread of the disease with 60-80% of cases developing liver lesionsduring the illness. The positive identification of liver metastasis istherefore a clear indication of latent disease. Although much lesscommon, other possible sites of spread include lung, brain andoccasionally bone.

Due to the strong correlation between extent of liver involvement(number and location of metastasis) and resectability, the appropriateevaluation of patients regarding suitability for surgery is becomingmore important (Liver Metastasis: Biology, diagnosis and treatment.Garden O. J., Gereghty J. G., Nagorney D. M. eds. 1998). The need for anagent capable of detecting metastasis of small size (<1 cm) will have aprofound impact on the treatment and management of CRC patients. Thereis a need to specifically detect small (<1 cm) metastatic lesions in theliver. The early detection of number and location of metastatic lesionsis critical. There is also a need to characterise and specificallyidentify the origin of the tumour with no interference from otherpossible lesions (e.g. cysts, benign lesions, non-treatable tumours).

A low-molecular weight heat-stable toxin is produced by enterotoxicstrains of E. coli. This toxin, known as ST peptide, mediates acutediarrheal disease by binding to its receptor on colorectal cells andstimulating guanylate cyclase. Synthetic ST peptides that bind to the STreceptor without mediating acute diarrheal disease are disclosed in U.S.Pat. No. 4,545,931 and U.S. Pat. No. 4,886,663. These syntheticallyproduced peptides are suitable for human administration for therapeuticand diagnostic purposes.

Targeted ligands directed towards receptors that are expressedselectively on tumour cells of colorectal origin are a means tospecifically detect the presence of cancers of colorectal origin. Thus,the ST receptor is a potential target mechanism. Gastrointestinalmucosal cells specifically express the ST receptor and the expressionpersists after colonic and rectal mucosal cells undergo malignantneoplasic transformation. No ST peptide has been found in any otherextra-intestinal tissues, therefore specificity of ligands to tissue ofgastrointestinal origin is maintained. Similar levels of expression havebeen found in human primary and metastatic colorectal tissues withdifferent grades of differentiation and location. A specific ligand forthe ST receptor will only bind to metastatic disease, as access to theapical side of intestinal cells will be avoided if the compound isinjected intravenously.

Radiolabelled ST peptides for CRC imaging and diagnosis have beenpreviously documented. U.S. Pat. No. 5,518,888 claims radiodiagnosticagents based on ST peptides. In one embodiment of that invention, thepeptides may be linked to a radioactive imaging agent, such asradioactive iodine, ¹¹¹In or ^(99m)Tc. ^(99m)Tc is chelated by DTPA,which is converted to an anhydride and reacted with an ST peptide. Noother chelates are disclosed in U.S. Pat. No. 5,518,888. Radiolabellingthus renders the peptides suitable for use in radioimaging metastasizedcolorectal cancers. U.S. Pat. No. 6,060,037 discloses a method ofradioimaging metastatic CRC using such radiolabelled ST peptides. Thedetection of localised accumulation or aggregation of radioactivityfollowing administration of radiolabelled ST peptide is indicative ofthe presence of cells with ST receptors. WO 99/21587 and WO 99/39748also disclose radiolabelled ST peptides for diagnostic imaging. In WO99/21587 the preferred classes of radiometal complexing agents areterpyridines and phenanthrolines. In WO 99/39748 the complexing agentscomprise a macrocyclic oligo-2,6-pyridine-containing ring which is aderivative of a terpyridine, quaterpyridine, quinpyridine, orsexipyridine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of pH 8.0, 8.5 and 9.0 in the radiolabellingreaction on the formation of Species 2 (CA-ST₅₋₁₈).

FIG. 2 shows the effect of varying the temperature of the radiolabellingreaction on the formation of Species 2 (CA-ST₅₋₁₈). The temperaturesevaluated were 40° C., 60° C. and 70° C.

FIG. 3 shows the effect of compound mass on the percent formation ofSpecies 2 (CA-ST₅₋₁₈), tested at 12.5 μg, 25 μg, 50 μg and 100 μgcompound.

FIG. 4 shows SPECT images of mice bearing T84 tumours subcutaneously.The images are planar posterior static images with LEUHR collimatorusing BPLC purified ^(99m)Tc-labelled CA1-ST₅₋₁₈ at 20 MBq per animal.Tumour to liver ratios were; 1.1 at 15 minutes, 2 at 60 minutes and 4 at120 minutes. Image quality was comparable when crude preparations wereused.

FIG. 5 shows the relative retention of CA1-ST₅₋₁₈ versus the negativecontrol, CA1-ST_(5-18(cys-ala)).

FIG. 6 shows a comparison of HPLC purified ^(99m)Tc-labelled CA1-ST₅₋₁₈and CA1-ST_(5-18(cys-ala)) uptake in CD-1 nude mice bearing subcutaneousT84 tumours. Planar posterior static imaging with LEUHR collimator usingHPLC purified CA1-ST₅₋₁₈ at 20 MBq per animal.

FIG. 7 shows the biodistribution of purified CA1-ST₅₋₁₈ at 2 MBq peranimal in RNU/mu nude rats and mice bearing T84 liver tumours. The datais expressed in as relative retention (RR) and ratios of tumour to liverand tumour to muscle.

FIG. 8 shows an image of an RNU/rnu nude rat bearing T84 liver tumours.It is a planar posterior static image at 120 minutes post-injection(p.i.) using HPLC purified CA1-ST₅₋₁₈ at 20 MBq with LEUHR collimator.The image is a whole body image with background counts removed andkidneys masked. Image tumour:liver ratio was typically 2.5.

FIG. 9 shows an image of RNU/rnu nude mice bearing T84 liver tumours inthe presence and absence of excess peptide. The images are planarposterior static images 120 minutes p.i. of BPLC purified CA1-ST₅₋₁₈ at20 MBq per animal with LEUHR collimator.

FIG. 10 shows the receptor density (RD) of two human and four xenografthuman cell line CRC tumours (nd=receptor density data not detected).

DESCRIPTION OF THE INVENTION

ST peptides are cyclic peptides containing three disulphide bonds. Ithas been found that these ST peptides are unstable under the basic pHconditions required for some ^(99m)Tc chelator radiolabelling, giving incleavage of the disulphide bridges and unacceptable loss of potency.This instability may rule out a large number of ^(99m)Tc chelates thatrequire pH ca. 8-9 for efficient labelling. Consequently,peptide-chelate conjugates would have to be labelled under acidicconditions to maintain high potency. However, such chelates needcomplicated labelling protocols and can generate ^(99m)Tc-conjugateswith poor efficacy, e.g., elevated hepatobiliary uptake and fast bloodclearance. The present invention provides peptide-chelate conjugatesthat can be labelled under basic conditions without loss of potency ofthe ST peptide, to provide effective imaging agents.

In a first aspect, the present invention provides a peptide-chelateconjugate comprising a peptide having affinity for the ST receptorconjugated to a tetradentate chelating agent, wherein the peptide havingaffinity for the ST receptor comprises 10 to 25 amino acids. “A peptidehaving affinity for the ST receptor” is a biologically active peptidewhich is suitably between 10 and 25 amino acids, and preferably between13 and 19 amino acids, derived from the ST peptide sequence, that bindsthe ST receptor with high affinity. The peptides of the presentinvention may be of naturally occurring or synthetic origin, but arepreferably synthetic. These peptides may be conjugated directly to thechelate, or by means of a “linker group”. “Linker groups” may comprise apeptide sequence of about 5 to 9 amino acids, with or without theinclusion of other groups such as aliphatic chains of up to 5 carbons inlength. Preferred linker groups are poly-Lys, poly-Glu, (Gly)₃-(DGlU)₃and (Gly)₃-(aminocaproic acid)₂. Labelled with a suitable radiometal, STpeptides of the invention are suitable for use as imaging agents todetect cancers of colorectal origin. Preferred peptides of the presentinvention are sequences 1-7.

Especially preferred peptides having affinity for the ST receptor areSEQ ID NOs 1 to 5, presented in the included sequence listing.

The negative control used in the experiments of the present invention isSEQ ID NO 8, i.e., SEQ ID NO 1 with all of the cysteines replaced withalanines and therefore lacking the disulphide bridges required forbinding.

As used herein the term “tetradentate chelating agents” are chelatesthat are suitable for the formation of the peptide-chelate conjugates ofthe present invention, in which the radiometal is coordinated by thefour metal donor atoms of the tetradentate chelating agent. A suitableradioactive metal ion is complexed by a tetradentate chelating agent bymeans of a donor set consisting of four metal donors which form at leastone 5- or 6-membered chelate ring with the radiometal. Preferably, thetetradentate chelating agent forms 2 or more such 5- or 6-memberedchelate rings with the radiometal. These ligands are particularlysuitable for the complexation of technetium (^(99m)Tc), but may also beused for other radiometals.

The tetradentate chelating agent is present in the conjugate in order toproduce a radiolabelled form of the peptide, suitable for diagnosticimaging. A suitable radioactive metal ion may be incorporated into thepeptide-chelate conjugate by means of complexation with the tetradentatechelating agent. It is an important feature of the present inventionthat the potency of the ST peptide is not compromised by the process ofradiolabelling.

Tetradentate chelating agents suitable for the present invention includebut are not limited to the following:

(i) diaminedioximes of formula A:

-   -   where R^(A1)-R^(A6) are each independently an R group;    -   where R is H or C₁₋₁₀ alkyl, alkylaryl, alkoxyalkyl,        hydroxyalkyl, fluoroalkyl, aminoalkyl or carboxyalkyl;    -   and Q is a bridging group of formula —(W)_(n)—;    -   where n is 3, 4 or 5 and each W is independently —O—, —NR— or        —CR₂— provided that (W)_(n) contains a maximum of one W group        which is —O— or —NR—.    -   Preferred diaminedioximes have R^(A1) to R^(A6)=C₁₋₃ alkyl,        alkylaryl alkoxyalkyl, hydroxyalkyl, fluoroalkyl or aminoalkyl.        Most preferred diaminedioximes have R^(A1) to R^(A6)=CH₃.    -   (ii) N₃S ligands of formula B:    -   where X is a thiol protecting group such as benzoyl, acetyl or        ethoxyethyl that is cleaved before or during the labelling        process, and;    -   R^(B1) and R^(B2) may be H or the side chain of any amino acid,

(iii) N₄ ligands of formula C:

-   -   where R^(C1)-R^(C4) may be H, alkyl, aryl or combinations        thereof and where one of R^(C1)-R^(C4) must be a functional        group such as alkylamine, alkyl sulphide, alkoxy, alkyl        carboxylate, alkyl arylamine or aryl sulphide. Ligands of this        type include those with C═O amide linkages. Macrocyclic versions        of formula C are also included, such as:    -   where R^(C5) is as defined for R^(C1) to R^(C4), above.

(iv) Diaminediphenols of formula D:

-   -   where Y is either C or N;    -   R^(D1) and R^(D2) may be either H, alkyl or aryl and where one        of R^(D1) and R^(D2) must be a functional group such as        alkylamine, alkyl sulphide, alkoxy, alkyl carboxylate, alkyl        arylamine or aryl sulphide, and;    -   m=n=1 or 2.

v) N₂S₂ ligands of formula E:

-   -   where Z is a thiol protecting group such as benzoyl, acetyl or        ethoxyethyl that is cleaved before or during the labelling        process, and;    -   R^(E1)-R^(E12) may be each chosen from H, an aryl group or an        alkyl group and where one of R^(E1)-R^(E) ¹² must be a        functional group such as alkylamine, alkyl sulphide, alkoxy,        alkyl carboxylate, alkyl arylamine or aryl sulphide, and;    -   one or more of the pairs R^(E3)/R^(E4), R^(E5)/R^(E6),        R^(E7)/R^(E8), R^(E9)/R^(E10) may represent a C═O bond.

A preferred chelating agent of the present invention is a diaminedioximeof formula A, where R^(A1)-R^(A6) are all CH₃, and Q is—(CH₂)₂NR(CH₂)₂—, where R is an R group as defined for Formula (A). Amost preferred chelating agent is where R of —(CH₂)₂NR(CH₂)₂— isaminoalkyl, especially R═—(CH₂)₂NH—. The latter will be referred to inthe remainder of this document as “chelating agent 1⇄ (CA1). Anotherpreferred chelating agent is an N₃S ligand of formula B where R^(B1) andR^(B2) are both H and X is acetyl. This will be referred to as“chelating agent 2” (CA2) for the remainder of the document. “Chelatingagent 3” (CA3) is an N₃S ligand of formula B where R^(B1) isCH₂CH₂CH₂C═O, R^(B2) is H and X is ethoxyethyl.

The synthetic peptides of the present invention can be synthesised bysolid phase methodology, as is well known in the art. A representativesynthesis of ST₅₋₁₈ is given in Example 2. The peptide-chelateconjugates of the present invention can be prepared using bifunctionalchelators, i.e., compounds in which the tetradentate chelator bears apendant fictional group. Preferred functional groups are amino orcarboxyl functional groups, which permit facile coupling via amide bondsto the amine or carboxyl groups on the peptides of interest, especiallythe amino- or carboxyl-terminus of the peptide.

N₃S bifunctional chelators can be prepared by the method of Sudhaker etal [Bioconjugate Chem., Vol. 9, 108-117(1998)]. The synthesis ofdiaminedioxime bifunctional chelators is described in Example 3.Diaminediphenol compounds can be prepared by the method of Pillai et al[Nucl. Med. Biol., Vol. 20, 211-216(1993)]. Bisamidedithiol compoundscan be prepared by the method of Kung et al [Tetr. Lett., Vol 30,4069-4072 (1989]. Monoamidemonoaminebisthiol compounds can be preparedby the method of Hansen et al [Inorg. Chem., Vol 38, 5351-5358 (1999)].Functional tetraamines can be prepared by the method of Simon et al [J.Am. Chem. Soc., Vol 102, 7247 (1980)]. The critical processes in thesynthesis of the CA1-ST₅₋₁₈ conjugate can be summarised as shown inScheme 1 on the following page.

Conjugation of the peptide to the chelate is carried out prior to thelabelling reaction. The direct method of conjugation is exemplified inthe compound CA1-ST₅₋₁₈ (or CA1-SEQ ID No 1). Conjugation via a linkermolecule is exemplified in the compound CA1-(Gly)₃-(D-Glu)₃-ST₅₋₁₈ (orCA1-SEQ ID No 5). Conjugations may be carried out via either theN-terminus or the C-terminus of the peptide molecule. Refer to Table Ifor these structures and structures of other suitable compounds of thepresent invention.

The compounds of the present invention have reproducibly highradiochemical purity (RCP), advantageous pharmacokinetics such asreduced gastrointestinal uptake, and imaging efficacy in tumouredanimals. The compounds offer a distinct advantage over previouslydisclosed CRC-targeting compounds since they have improvedpharmacokinetics and specific retention in tumour tissue compared tobackground tissues.

In a second aspect, the present invention provides a metal complexcomprising a radiometal complexed to the tetradentate chelating agent ofthe peptide-chelate conjugate. Preferred chelating agents for the metalcomplex are chosen from diaminedioximes of formula A where R^(A1)-R^(A6)are all CH₃ and Q is —(CH₂)₂NR(CH₂)₂—. An especially preferreddiaminedioxime of formula A is CA1, as defined above. Other preferredchelating agents for the metal complex are the N₃S ligands CA2 and CA3,defined above. A suitable radiometal may be chosen from positronemitters such as ⁶⁴Cu and ⁶⁵Cu, or from gamma emitters such as ^(99m)Tc.A preferred embodiment of the invention is a peptide-chelate conjugateradiolabelled with ^(99m)Tc. A peptide-chelate conjugate radiolabelledwith ^(99m)Tc, where the radiolabelling reaction is carried out at basicpH is a most preferred embodiment of the present invention.

Radiolabelling as defined by the present invention is the chemicalattachment of a radiometal, such as those described above. Suitableradiolabelled compounds of the invention have affinity for the STreceptor to enable diagnostic imaging of cancers of colorectal origin.Preferred radiolabelled compounds are thus cleanly labelled with theminimum production of by-products. An example of such a by-product isreduced hydrolysed technetium (RHT) in the case of ^(99m)Tc labelling.In a preferred embodiment of the invention, the peptide-chelateconjugate is labelled with ^(99m)Tc maintaining high RCP and low RHTlevels. The results of various studies described in the Examples showthat the preferred compounds of the present invention are stable to thelabelling conditions and maintain their biological efficacy followingradiolabelling.

The RHT levels are of particular importance in the case of^(99m)Tc-labelled conjugates of the present invention as one of the mainbiological targets is in the liver. RHT localises in the liver and thusif allowed to remain, will therefore increase background tissueactivity, decrease target to background ratios and lead to an overalldecrease in image quality. The amount of RHT is relatively low for allthe preparations (Example 10). There is little difference between thelabelling characteristics of CA1-ST₅₋₁₈ at conjugate levels of 12.5-100μg, in terms of the rate of formation of species 2. However, there wasan increase in RHT at reduced conjugate levels (Example 10).

Compared to the non-HPLC purified unfiltered preparation, both filteringand addition of methylene diphosphonate (MDP, which is known to reduceRHT levels) had a similar effect in reducing liver uptake (Example 24).Neither method reduced background tissue counts to the level seen withHPLC purified preparations but high quality images of liver metastasesare still possible with non-HPLC purified preparations.

Radiolabelling of the peptide-chelate conjugate may be carried out in asuitable buffering system such as carbonate, borate, triethanolaminehydrochloride-NaOH, dimethylleucylglycine,tris(hydroxymethyl)aminomethane, 2-amino-2-methyl-1:3-propanediol-HCl,diethanolamine-HCl, Clark and Lubs solutions (Bower and Bates, J. Res.Nata. Bur. Stand. 55, 191 (1957), Glycine, Glycylglycine or TAPS®. Thelabelling reaction is preferably carried out at a basic pH, i.e.,between pH 8.0 and pH 10, most preferably at a pH of around 8.5.Examples 5 and 6 describe conducting the radiolabelling reaction usingeither sodium hydroxide or borate buffer in order to maintain a suitablepH. A preferred method of the present invention is the use of a boratebuffering system in the radiolabelling reaction.

The competition binding experiments carried out (as described in Example15) investigated the relative potency of various ST peptide compounds.Ki values in the nM range suggest good binding of competing ligand.These data show firstly that variations outside the pharmacophore in theST peptide make little difference to binding (ST₁₋₁₇ vs. ST_(5-18,) Ki0.6 and 0.4 nM, respectively). The presence of CA3 on the ST peptideresults in a small increase in Ki, i.e., a small resulting decrease inbinding. The presence of CA1 has a greater effect on binding to reducefurther the Ki value to 2.8 nM, almost a ten fold reduction in bindingefficacy. Binding affinity in the nanomolar range is consideredacceptably high and the resulting differences in reduced binding are notobserved in vivo. The negative control demonstrated no binding (up to500 μM).

The preferred compounds of the present invention demonstrated Ki valuesof less than 100 nM, indicating high affinity for the receptor (seeExample 17). Of all the compounds tested, CA1-ST₅₋₁₈, CA3-ST₅₋₁₈ andCA1-gly₃-Dglu₃-ST₅₋₁₈ showed the greatest potency in this assay, with Kivalues of 2.8 nM, 0.8 nM and 1.0 nM, respectively. The in vivo efficacyof these screened compounds is discussed below.

The data obtained in Example 15, and the high binding (Ki) ofmock-labelled peptide (peptide subjected to the radiolabelling protocolin the absence of radiometal), supports the fact that the radiolabelledmaterial is still bioactive.

In a third aspect, the invention also encompasses radiopharmaceuticalpreparations. By the term “radiopharmaceutical preparation” is meant acomposition comprising the radiometal complex of the invention in a formsuitable for human administration. For human administration aradiopharamceutical preparation must be sterile. It is preferably in aninjectable form, e.g., for ^(99m)Tc, reconstitution of thepeptide-chelate conjugate with sterile pertechnetate in saline.

The radiopharmaceutical preparation of the present invention may also beprovided in a unit dose form ready for human injection and could forexample be supplied in a pre-filled sterile syringe. The syringecontaining the unit dose would also be supplied within a syringe shield(to protect the operator from potential radioactive dose).

A fourth aspect of the present invention is the use of theradiopharmaceutical preparation of the invention for the imaging ofcancer of colorectal origin. Imaging using the radiopharmaceuticalpreparation of the present invention may be carried out by means of PETor SPECT imaging, depending on the nature of the radiometal.

In a fifth aspect, the present invention provides kits for theproduction of the radiopharmaceutical preparation of the invention.Suitable kits are designed to give sterile radiopharmaceutical productssuitable for human administration, e.g. via injection into thebloodstream. When the radiometal is ^(99m)Tc, the kit comprises a vialcontaining the peptide-chelate conjugate for the metal together with apharmaceutically acceptable reducing agent. Suitable such reducingagents are: sodium dithionite, sodium bisulphite, ascorbic acid,formaridine sulphinic acid, stannous ion, Fe(II) or Cu(l). Thepharmaceutically acceptable reducing agent is preferably a stannous saltsuch as stannous chloride or stannous tartrate. Alternatively, the kitmay comprise a metal complex which, upon addition of the radiometal,undergoes transmetallation (i.e., ligand exchange) giving the desiredproduct. For ^(99m)Tc, the kit is preferably lyophilised and is designedto be reconstituted with sterile ^(99m)Tc-pertechnetate (TcO₄ ⁻) from a^(99m)Tc radioisotope generator to give a radiopharmaceutical solutionsuitable for human administration without further manipulation.

The peptide-chelate conjugate is preferably present in the kit in a formamenable to transport and storage over relatively long time-periods.Reconstitution with eluate from a ^(99m)Tc generator under the preferredradiolabelling conditions results in a ^(99m)Tc-labelled peptide withspecificity for the ST receptor. It is an important feature of thepresent invention that in such a kit, the radiolabelling reaction doesnot alter the affinity of the peptide for the ST receptor.^(99m)Tc-labelled peptides of the present invention have been shown tobe stable for up to 4 hrs at room temperature and in the presence ofactivity levels of up to 1 GBq.

The above kits or pre-filled syringes may optionally contain furtheringredients such as buffers; pharmaceutically acceptable solubilisers(e.g. cyclodextrins or surfactants such as Pluronic™, Tween™ orphospholipids); pharmaceutically acceptable stabilisers,radioprotectants or antioxidants (such as ascorbic acid, gentisic acidor para-aminobenzoic acid) or bulking agents for lyophilisation (such assodium chloride or mannitol).

It is a feature of the invention that the metal complexes havemaintained potency for the ST receptor as well as suitablephannacokinetics. Data obtained in Example 16 indicate that positivecontrol, ¹²⁵I-ST-₁₋₁₇ has the highest binding of 20% of addedradioactive material. The negative control, CA3-T_(5-18(Cys-Ala)),showed no specific binding (binding at NSB level, <1%).^(99m)Tc-labelled CA1-STs₅₋₁₈ shows reduced binding (6% of total added)compared to the positive control but is still significantly higher thanthat of the negative control. The compounds of the present inventionalso have optimal blood clearance, fast liver clearance and fastbackground tissue clearance. Liver clearance is a particularlysignificant feature of the present invention due to the high incidenceof CRC metastases in that organ. Furthermore, the compounds of thepresent invention have been demonstrated to permit imaging of colorectalcancer metastases which are less than 1 cm in diameter. This is afeature which additionally permits distinction to be drawn between focaland diffuse metastases. It has not been previously possible to achievethese features in an agent directed towards the ST receptor. This istherefore a surprising development over the prior art.

Metal complexes of the present invention are cleared rapidly via theurine (>90% at 60 min p.i.). An additional feature of the presentinvention is low gastrointestinal uptake, which reduces the risk ofunwanted side effects. Low uptake in background tissues such as liver,which may interfere with image quality, is an advantage of the metalcomplexes of the present invention. For CA1-ST₅₋₁₈ the liver uptake was0.93% of the injected dose per gram at 2 hours p.i. (Example 17).CA1-ST₅₋₁₈ shows the greatest tumour to background tissue ratio, thegreatest uptake (% ID/g) and relative retention of all the compoundsassessed. This shows that of all the compounds examined, CA1-ST₅₋₁₈showed the best pharmacokinetic qualities for a CRC imaging agent(Example 17).

The preferred mode of imaging of the present invention is SPECT imaging.It was demonstrated to be possible to image tumours of between 0.5-1 cmin diameter by SPECT imaging using the subcutaneous model (described inExample 17). The images acquired in Example 18 show that imaging ispossible from 15 min p.i., and by 120 min p.i. only the tumour andkidneys are visible. The tumour size imaged was of the region expectedin the target population. The images have been acquired in a planarmode, with a relatively low radioactive dose of 20 MBq/animal. Spatialresolution between organs is known to be better in humans such that theimages obtained would be expected to be at least as good as seen in theanimal models.

As shown in Examples 18, 19, 21 and 23, the preferred compounds of theinvention display superior imaging properties not previously reported inthe art. Specific in vivo tumour uptake has been demonstrated in twodifferent animal models. These models are described in Examples 17 and20. Imaging of both subcutaneous tumours and liver metastases wasachieved at <2 hr p.i. in mice. It was possible to acquire images inmice from 15 minutes p.i. This will support the ideal clinical imagingwindow in humans of between 1-6 hours p.i.

The images produced in Example 18 (FIG. 4) have consistently shown clearidentification of tumours from 15 min p.i. with good ratios for tumourto muscle (not shown) and tumour to liver, which improves over time sothat by 120 min p.i. only the tumour and kidneys are visible. The dataobtained in Example 19 demonstrate good uptake for ^(99m)Tc-labelledCA1-ST₅₋₁₈ and poor uptake for the negative control (FIG. 5). Planarposterior static images of the same animals acquired at 120-min p.i.show a similar pattern; good image of ^(99m)Tc-labelled CA1-ST₅₋₁₈ andpoor image of negative control (FIG. 6). In the image produced inExample 21 (FIG. 8), metastases are clearly visible, with a tumour:liverratio of 1.5-3.0 based on ROI analysis. Images were acquiredconsistently and without major manipulation, with both of theformulations: HPLC purified and crude preparations.

Imaging experiments as described in Example 21 demonstrate specificretention of ^(99m)Tc-labelled CA1-ST₅₋₁₈ into CRC tumours. These datasupport the specificity of ^(99m)Tc-labelled CA1-ST₅₋₁₈ forST-receptors, and highlight the importance of the conformationalstructure of ST peptides. When mice bearing sub-cutaneous Lewis lungtumours were injected with CA1-ST₅₋₁₈, as described in Example 22,significantly higher uptake of CA1-ST₅₋₁₈ into colorectal tumours thaninto those of non-colonic origin was demonstrated.

Literature studies indicate little variation in receptor expressionbetween tumour samples, and the reported data (80-120 fmol/mg protein)compared well with the average expression found in this study. Althougha large differential in receptor expression has been seen between thetwo human donors (Example 25)

In biodistribution studies on a range of xenograft tumours withdifferent ST receptor expression, highest expression was shown in T84cells but similar to the expression in human tumours (Example 25). Therelative expression in a number of different cell lines was analysed andwas found to be in the following order: T84>CaCO2>LS180>HT29>SW480. Thedata show comparatively higher retention of CA1-ST₅₋₁₈ in high STreceptor-expressing tumours, CaCO2 and T84, compared to the rest. Imagesof tumours expressing receptor density lower than that found in humantumours (LS180) was achieved, as a result CA1-ST₅₋₁₈ will be able todetect a wide range of human tumours.

Expected values from previous studies suggest little loss in expressionwith either origin of tumour or decrease in grade. Therefore, apopulation of patients with low ST receptor-expressing metastases isunlikely. More importantly, the expression of receptors in human CRCtumours lies directly in between that of two imageable CRC tumours,suggesting that expression of the receptor in Human tumours issufficient for imaging with ^(99m)Tc-labelled CA1-ST₅₋₁₈. Low retentionin tumours was observed with the negative control,CA3-ST_(5-18(cys-ala)). The data also show that despite very lowreceptor density there is still specific retention of ^(99m)Tc-labelledCA1-ST₅₋₁₈ in those tumours. The tumour uptake in tumours from T84 andLS180 cells, grown subcutaneously or in the liver of immunocompromisedanimals, is sufficient for imaging, as demonstrated in both thesubcutaneous model and in the liver metastases model. By doing so it hasbeen shown that it is possible to image small tumours in the region ofexpression levels of ST receptor found in human tumours.

The compounds of the present invention satisfy all major requirementsidentified for an improved CRC imaging agent, in particular successfullyimaging tumours in an animal model thought to represent the morechallenging aspects of the clinical disease. Exceptional efficacy hasbeen demonstrated in the critical efficacy model of liver metastases.The preferred embodiments allow imaging of tumours that represent therange of receptor densities found in human liver metastases from CRC.The efficacy is maintained in the presence of excess peptide, at levelsexpected in a potential clinical formulation hanging studies have shownthat preferred compounds are likely to be more sensitive and specificthan X-ray CT. The compounds of the present invention could thus be usedin the initial diagnosis of disease, and more importantly in theappropriate staging or determination of the extent of the disease,measuring response to therapy and in the follow up of patients treatedfor primary CRC.

The compounds of the present invention are therefore superior to anyother reported CRC imaging compound. They are retained specifically intumours of CRC origin. Imaging potential was demonstrated in aclinically acceptable time frame in a desired target tumour size.Surprising uptake, retention, pharmacokinetics, and efficacy have beendemonstrated in view of reported instability of the vector at basic pH.

EXAMPLES Example 1 Peptide-Chelate Conjugates

The compounds used are given in Table I. Conjugates were prepared asdescribed in Example 2 as 100 μg aliquots in plastic vials, and storedat −18° C. The conjugates were allowed to warm to ambient temperaturebefore use. TABLE I Compounds of the invention CA3-ST₅₋₁₈

CA3-ST_(5-18 (cys-ala))

CA1-ST₅₋₁₈

CA₂-(Gly)₂-Glu-(Lys)₃-ST₅₋₁₈

CA₂-(Gly)₂-Glu-Lys-Glu-Lys-ST₅₋₁₈

ST₅₋₁₇ C terminus amide

CA2-(Phe)₂-(CH₂)₅-ST₅₋₁₈

CA1-(Glu)₆-ST₅₋₁₈

CA1-(Lys)₆-Gly-ST₅₋₁₈

CA1-(Gly)₃-(D-Glu)₃-ST₅₋₁₈

CA1-(Gly)₃-(aminocaproic acid)₂-ST₅₋₁₈

Example 2 Synthesis of CA1-ST₅₋₁₈

2.1 Solid-Phase Synthesis

The solid-phase assembly of the peptide sequence was performed on thesupport Fmoc-Tyr(tBu)-SASRIN (10 g polymer; 0.6 mmol/g loading) using anAdvanced Chemtech 90 automated peptide synthesiser. The amino acids wereall of the L-configuration with the α-amino function blocked with the9-Fluorenylmethoxycarbonyl (Fmoc) protecting group.

The reactive side-chain functional groups were bearing the followingprotecting groups:

-   -   Trityl (Trt)—for cysteines 6 and 14 and asparagine,    -   Acetamidomethyl (Acm)—for cysteines 5 and 10,    -   4Methoxybenzyl (Mob)—for cysteines 9 and 17,    -   tert-Butyl ester—for glutamic acid    -   tert-Butyl ether—for tyrosine        2.2 Coupling and Deprotection Reactions

Double coupling cycles were carried out using a 5-fold excess ofactivated Fmoc-amino acid derivatives dissolved in N-methylpyrrolidone(NMP). The first coupling was carried out with Fmoc amino acid/DIC/HOBtactivation, and the second coupling with Fmoc amino acid/HBTU/HOBt/DIEAactivation.

Excess reagents were removed from the polymer by washing three timeswith NMP, three times with methanol and three times with NMP beforeremoval of the Fmoc group in 20% piperidine/NMP (5 minute and 20 minutecycles performed). Once assembled the peptide resin was washed threetimes with NMP, three times with DCM and three times with methanolbefore drying overnight in a vacuum oven at room temperature. Weight offinal resin 20.76 g (yield 84.2%).

2.3 Cleavage of Partially Protected Peptide from the Solid Support

To the dry peptide resin in the nitrogen bubbler was added TFA (25 ml)containing 5% TIS and 5% water and the mixture bubbled under nitrogenfor 30 minutes. The peptide solution was then drained into toluene (200ml) and evaporated in vacuo at RT. Cold diethyl ether was added to theoily residue in order to precipitate out the product as a solid.Following trituration with diethyl ether and drying, 600 mg of the crudedithiol peptide was obtained. The HPLC purity of the crude product wasshown to be around 70% with several side-products identified due tomodifications during the TFA cleavage step. The most significantimpurity (10-15%) was caused by the cleavage of one of the CA1 oximeside arms.

2.4 Cyclisation 1: Oxidation of the Crude Dithiol Peptide: 6,14Disulphide

Crude dithiol peptide from 2.3 above (0.132 mmol, 300 mg) was dissolvedportionwise in 600 mL of 0.1M NH₄HCO₃ (pH 8) in water/acetonitrile(80:20) containing potassium ferricyanate (0.3 mmol, 100 mg). Thereaction was left stirring under nitrogen for 16 hours then HPLCanalysis was used to confirm complete conversion of starting dithiol tooxidised product. The peptide solution was then acidified to pH 2 withTFA, filtered through a 0.45 micron filter (Millipore) then pumpeddirectly onto the preparative HPLC column and gradient elutioninitiated. Fractions of >80% purity were combined and freeze-driedyielding 140 mg of the desired product.

2.5 Cyclisation 2: Acm Group Deprotection and Cyclisation 6,14: 5,10

The mono-disulphide product from 2.4 above (0.14 g, 0.062 mmol) wasdissolved in 150 ml of acetic acid/DMSO (1:1) containing 0.1 mL anisoleand iodine (0.1 g, 0.39 mmol) added. The reaction mixture was stirred inthe dark for 30 minutes before dilution to a total volume of 450 ml withdistilled water. The peptide solution was then extracted twice withdiethyl ether in a separating funnel and the aqueous phase analysed byHPLC.

BPLC analysis revealed the presence of two new products present in a 4:1ratio. Both products had the desired molecular weight as evidenced by MSanalysis and corresponded to two conformational isomers. The aqueouslayer was once again filtered through a 0.45 micron filter (Millipore)before pumping directly onto the preparative BPLC column and gradientelution initiated. Fractions of >80% purity were combined andfreeze-dried yielding 40 mg of the desired product.

2.6 Cyclisation 3: Mob Group Deprotection and Cyclisation

To the bis-disulphide product from 2.5 above (40 mg, 0.019 mmol) wasadded a solution of 10% DMSO/TFA (50 ml) containing 0.1 ml of anisoleand the mixture gently shaken (20 min.). The TFA was evaporated in vacuoat room temperature and the product precipitated by the addition ofdiethyl ether. Following trituration with diethyl ether 25 mg of thefully folded product was recovered. Preparative HPLC of crude productand freeze-drying yielded 16 mg of the desired product (purity >98%). MSanalysis by LCQ; found (M+H)+=1873, expected (M+H)+=1873.

2.7: Salt Exchange

The pure TFA salt from above was dissolved in water containing 0.1%ammonium acetate. Gradient elution on a C18 reverse phase column using a10 to 60% B gradient over 40 minutes where buffer A=0.1% ammoniumacetate/H₂O; B=0.1% ammonium acetate in 80% acetonitrile/H₂O) wasperformed. The acetate salt (15 mg) was recovered followingfreeze-drying.

Example 3 Synthesis of Chelating Agent 1 (CA1)

To a solution of tris-(2-aminoethyl) amine (Aldrich; 2 ml, 13 mmol) inacetonitrile (20 ml) was added sodium bicarbonate (2.2 g, 26 mmol). Asolution of 3-chloro-3-methyl-2-nitrosobutane (1.8 g, 13 mmol) in dryacetonitrile (10 ml) was added slowly at 0° C. The reaction mixture wasleft to stir at room temperature for 4 hours, and then filtered. Thefiltrant was washed with acetonitrile and the filtrate evaporated. Thecrude product was dissolved in acetonitrile and purified by HPLC(Hamilton PRP-1; A: 2% aqueous NH₃, B: MeCN; 0-100% B in 20 min; 3ml/min) to afford CA1. Yield: 0.88 g, 19%.

Example 4 Conjugation of Peptide to Chelating Agent

4.1 Coupling of Succinic Acid Linker

An aliquot (1 g, ca 0.3 mmol) of the peptide resin from Example 2 wastransferred to a nitrogen bubbler where a further set of washes in DMF(3×15 ml) were carried out. A solution containing 1 mmol of succinicanhydride dissolved in DMF (20 ml) was then added and the mixturebubbled under nitrogen for 30 minutes. The polymer was washed with 5×15ml quantities of DMF and a resin sample taken for analysis by KaiserTest to confirm the absence of free amino groups.

4.2 Coupling of Chelating Agent 1 (CA1)

The resin bound acid functionality was then pre-activated in situ by theaddition of a solution in DMF (15 ml) containing PyAOP (0.52 g, 1 mmol),HOAt (0.14 g, 1 mmol) and NMM (0.2 ml, 2 mmol). On-resin activation wascarried out for 10 minutes followed by addition of a solution in DMF (10ml) of CA1 (0.4 g, 1.1 mmol). The mixture was bubbled for 3 hours thenexcess reagents removed by filtration followed by washing with DMF (5×15ml), DCM (3×15 ml) and ether (3×15 ml). The peptide-resin was then driedin a stream of nitrogen.

Example 5 Radiolabelling: Sodium Hydroxide Method

An aliquot of conjugate (100 μg) was dissolved in nitrogen-purged saline(900 μl) and added to a silanised P6 vial using silanised pipette tips.The pH of the solution was adjusted to pH 8.5 with 0.01M sodiumhydroxide solution and the vial capped. To the solution was addedNa^(99m)TcO₄ (1 ml, 1 GBq/ml) from a freshly eluted generator (eluateless than 2 hours old). The pH of the solution was again adjusted to pH8.5 with 0.01M sodium hydroxide solution. Freshly preparednitrogen-purged SnCl₂ solution (0.1 ml, 10 μmg/100 μml saline) was thenadded to the solution. The pH of the solution was again adjusted to pH8.5 with 0.01M sodium hydroxide solution. The vial was shaken after eachaddition to ensure mixing. The preparation was left to stand at roomtemperature.

Example 6 Radiolabelling: Borate Buffer Method

12.5 mM borate buffer (1 ml) was added to an aliquot of conjugate andsonicated for 30 seconds. The resulting solution was added to asilanised P6 vial, using silanised pipette tips, and the vial capped. Tothe solution was added Na^(99m)TcO₄ (1 ml, 1 GBq/ml) from a freshlyeluted generator (eluate less than 2 hours old). Freshly preparednitrogen-purged SnCl₂ solution (0.1 ml, 10 mg/100 ml saline) was thenadded to the solution. The vial was shaken after each addition to ensuremixing.

Example 7 Labelling Analysis

Investigation of the labelling characteristics of the CA1 conjugates wasperformed by labelling CA1-ST₅₋₁₈ using the sodium hydroxide methodoutlined in Example 5. Simultaneous BPLC and ITLC analysis was performedat approximately 15, 60 and 120 minutes post reconstitution. Inaddition, preparative HPLC was performed to obtain pure samples of thetwo ^(99m)Tc species. ITLC analysis was performed on each purifiedsample to confirm agreement between the two analytical techniques.Further studies were performed on alternative CA1 conjugates to confirmthe observed labelling characteristics.

The results of the initial investigation of the labellingcharacteristics showed a single major ^(99m)Tc species at early timepoints, converting almost completely to a second species over a numberof hours. Good agreement between ITLC and BPLC analysis was observed.Two ^(99m)Tc-conjugate species were resolved by both BPLC and ITLC andthe relative quantities of each species correlated well. For furtherconfirmation, preparative “PLC was performed to obtain pure samples ofthe two species.

Example 8 Effect of pH on Radiolabelling

The effect of pH on the labelling characteristics was investigated bycomparing the labelling of CA1-ST₅₋₁₈ (100 μg), using the borate buffermethod outlined in Example 6, at pH 8.1, 8.5 and 9.0. ITLC analysis wasperformed at 15, 30, 60, 90, 120 and 180 minutes post-reconstitution. Arelatively narrow pH range was studied due to the known instability ofthe ST₅₋₁₈ vector to high pH, caused by the presence of three disulphidebridges.

The results of the comparison of the labelling of CA1-ST₅₋₁₈ at pH 8.1,8.5 and 9.0 are shown (FIG. 1). The results are expressed as % species 2formed, as seen by ITLC (Gelman ITLC/SG paper, 70:30 saline:acetonitrile eluent) analysis.

Example 9 Effect of Temperature on Radiolabelling

The effect of temperature on the labelling characteristics wasinvestigated by comparing the labelling of CA1-ST₅₋₁₈ (100 μg), usingthe borate buffer (pH 9.0) method outlined above, followed by heating at40, 60 and 75° C. Each preparation was allowed to stand at roomtemperature for 10 minutes post reconstitution then heated for 20minutes. ITLC analysis was performed prior to heating (10 minutes postreconstitution), 10 minutes post heating (40 minutes postreconstitution) and 60 minutes post heating (100 minutes postreconstitution).

The results of the comparison of the labelling of CA1-ST₅₋₁₈ at 40, 60and 70° C. are shown (FIG. 2). The results are again expressed as % Peak2 formed, as measured by ITLC (Gelman ITLC/SG paper, 70:30 saline:acetonitrile eluent) analysis.

Example 10 Effect of Conjugate Level on Radiolabelling

The effect of conjugate level on the labelling characteristics wasinvestigated by comparing the labelling of various amounts of CA1-ST₅₋₁₈(12.5, 25, 50 and 100 μg) using the borate buffer (pH 9.0) methodoutlined above. ITLC analysis was performed at 15, 30, 60, 90, 120 and180 minutes post reconstitution.

The results of the labelling CA1-ST₅₋₁₈ at varying levels (12.5, 25, 50and 100 μg, equivalent to 6.6×10⁻⁹, 1.3×10⁻⁸, 2.7×10⁻⁸ and 5.3×10⁻⁸moles) are shown (FIG. 3). The results are expressed as the percentageof species 2 formed, as seen by ITLC (Gelman ITLC/SG paper, 70:30saline: acetonitrile eluent) analysis. RHT data has been shown (TableII). TABLE II RHT levels in radiolabelled preparations. 100 μg 50 μg 25μg 12.5 μg RHT (%) 0.6 1.5 1.9 2.7

Example 11 Stability of Conjugate to Labelling Conditions

To establish the stability of the starting material to the labellingconditions, an inactive, “blank” labelling experiment was performed. A“blank” labelling involved the substitution of saline for Na^(99m)TcO₄.CA1-ST₅₋₁₈ (100 μg) was subjected to the borate buffer labelling method(pH 9.0, 60° C., 20 minutes). The starting material was then HPLCpurified and a competition binding assay performed to determine whetherthere was any loss of affinity for the receptor compared with before thelabelling procedure. HPLC analysis was performed to detect anydegradation of the compound.

No degradation of the compound was observed by HPLC and potency(measured by competition binding assay) did not significantly alter.

Example 12 Competition Between CA1-benzamide and ST₅₋₁₈

To investigate co-ordination of ^(99m)Tc to the peptide conjugate, acompetition study was performed. Firstly, CA1-benzamide (which waschosen to mimic the CA1 portion of CA1-ST₅₋₁₈) and ST₅₋₁₈ were labelledindependently using the borate buffer method (pH9.0, 60° C. for 20minutes) and characterised by BPLC and ITLC. For the competition study,equimolar quantities of CA1-benzamide (31 μg, 6.92×10⁻⁸) and ST₅₋₁₈ (100μg, 6.92×10⁻⁸) were labelled together in the same pot using the boratebuffer method. ITLC and HPLC analyses, as outlined in Examples 13 and14, determined where the technetium had coordinated.

Example 13 ITLC

Gelman ITLC/SG paper eluted with saline

Gelman ITLC/SG paper eluted with methylethyl ketone (MEK, 2-butanone)

Gelman ITLC/SG paper eluted with a 70:30 v/v mixture of saline andacetonitrile

Whatman No. 1 (W1) paper eluted with a 50:50 v/v mixture of water andacetonitrile

Example 14 HPLC

All analysis was carried out using Gilson hardware and Unipointsoftware.

Column: Phenomenex Luna C-18, 4.6×250 mm, 5 μm

Eluent A:0.1% acetic acid (pH raised to pH 5.0 with ammonia) in 90%water/10% acetonitrile

Eluent B:0.1% acetic acid (pH raised to pH 5.0 with ammonia) in 10%water/90% acetonitrile

Flow:1 ml/min

UV Detector:)λ=230 mn

Similar gradients were used for the different conjugates. A typicalgradient was:

T₀:0% B

T₅:15% B

T₂₀:15% B

T₂₁:0% B

T₃₀:0% B

Example 15 Competition Binding Assay

Compounds were assessed for their potency by radioligand binding assayto determine inhibition constant (Ki) as a measure of potency ofbinding. Methods are described elsewhere, but briefly, [¹²⁵]]ST-₁₋₁₇ wascompeted with cold ST compounds (0.0001-50 μM) in 96 well platescontaining ca. 1 μg rat intestinal membrane and incubated 1 hr, 37° C.Samples were filtered (GF/B filters, Whatman) to retrieve boundradiolabelled peptide and counted to determine % Bound/Free.

Potency of compounds was determined by inhibition constant (Ki) fortheir ability to compete with radioligand, [¹²⁵I]ST₁₋₁₇. Potencies ofscreened compounds are shown in Table III. TABLE III Ki assay data forCRC compounds. Ki values shown are means of triplicate values of assaywells. Non-specific binding accounts for less than 0.1% in all cases andtotal binding (no competing peptide) is no greater than 20%. CompoundMean Ki value (nM) Comments ST₁₋₁₇ 0.6 ST peptide ST₅₋₁₈ 0.4 SelectedCore Vector CA3-ST₅₋₁₈ 0.8 Labelled CA3-ST_(5-18(cyc-ala)) none NegativeControl CA1-ST₅₋₁₈ 2.8 Unlabelled CA-ST₅₋₁₈ 3.5 Mock-labelled

Example 16 Radioactive Binding Assay

To determine whether radiolabelling affected the binding of thecompound, labelled compounds were tested for their ability to bind thereceptor. Briefly HPLC purified radiolabelled compounds (ca. 0.05 nM)were incubated with 6 μg/150 μl final volume of rat intestinal membrane(one hour), in the presence and absence of competing peptide (50 μMST₅₋₁₈). Samples were filtered (GF/B filters, Whatman) to retrieve boundradiolabelled peptide and counted to determine % Bound/Free.Non-specific binding accounted for less than 1% in all cases.

In order to ensure that potency was maintained post-labelling, an assaymeasuring binding of labelled compounds was developed.

Example 17 Biodistribution in a Subcutaneous Tumour Model

The sub-cutaneous model was used as an appropriate initial in vivoscreen. Briefly, mice (female nude CD-1, ca. 20 g; Charles River) wereinjected sub-cutaneously into the neck with T84 human colon carcinomacells (0.1 ml, 1×10⁸ cells/ml in medium) through a 23-gauge needle andallowed to develop tumours over 6-8 weeks.

At least three animals per test point were studied. Results areexpressed as % injected dose per gram (% ID/g) of tissue. Due to thefast clearance of ^(99m)Tc-labelled CA1-ST₅₋₁₈ (greater than 90%excreted via urine 60 mins p.i.) tumour uptake is also expressed asrelative retention (RR).

Compounds were screened for their uptake into sub-cutaneous T84 tumoursin the mouse (Table M). This model allows assessment of the imageabilityof CA1-ST₅₋₁₈ by allowing comparison between tumour and backgroundtissue uptake. TABLE IV Screening data for compounds of the invention.Screen CA3-ST_(5-18(cys-ala)) CA3-ST₅₋₁₈ CA1-ST₅₋₁₈ CA1-lys₆-gly-ST₅₋₁₈CA1-gly₃-Dglu₃-ST₅₋₁₈ Ki value (nM) none 0.8 2.8 72 1 HBS 26 5 1.0 1.480.71 (% id at 2 hours p.i.) Urinary (% id at 2 32 90 >90 76 63 hoursp.i.) Liver (% id at 2 hours p.i.) 0.93 0.5 0.1 4.9 0.46 Tumour uptake &ratios % id/g at 2 hours p.i.) 0.21 0.8 0.94 1.0 0.26 RC at 2 hours p.i.0.1 0.24 0.32 0.3 0.08 RR at 2 hours p.i. 0.17 2.5 6.4 1.6 1.9Tumour/liver 0.08 2 12.4 0.23 0.63 Retention over 2 hours 8 30 20 34 5.5

Example 18 Imaging in the Subcutaneous Tumour Model

Tumours were grown as in Example 17. Animals were monitored for tumourgrowth for 8 to 10 weeks, until tumours were 0.5-1 cm in size. Afterthis time, animals were injected with test article (0.1 ml, 20 MBq/ml or200 MBq/ml) as an intravenous bolus via the tail vein. At various timesp.i. animals were euthanased and whole body planar images acquiredbetween 5 minutes and 24 hours p.i. using a gamma camera (Park MedicalIsocam-1). Image acquisition times were typically 15-30 min or 150-250Kcounts (whole body, LEUHR collimator).

Example 19 Uptake of ^(99m)Tc-Labelled CA1-ST₅₋₁₈ andCA3-ST_(5-18(cys-ala)) (Negative Control) into CRC Tumours

CA3-ST_(5-18(cys-ala)) has the same peptide sequence as^(99m)Tc-labelled CA1-ST₅₋₁₈, except all cysteines have been replaced byalanine amino acids, removing the three disulphide bridges required forbinding. CA3-ST_(5-18(cys-ala)) also contains the CA3 as opposed to CA1and so was labelled using a suitable protocol for this chelate. Toexamine the uptake and retention of CA3-ST_(5-18(cys-ala)) and^(99m)Tc-labelled CA1-ST₅₋₁₈, CD-1 nude mice bearing sub-cutaneous T84tumours were injected, euthanased and dissected at various times p.i.The data is presented in FIGS. 5 and 6.

Example 20 Biodistribution in Liver Metastases Model

The liver metastases model was used in both mice and rats. As for thesubcutaneous tumour model except in establishing tumours, cells wereinjected via the spleen and allowed to transfer to the liver via thecirculation. Two minutes after injection of cells, a splenectomy wasperformed. Animals were allowed to generate tumours over 6-8 weeks.

Animals were monitored for tumour growth for 8 to 10 weeks, untiltumours were 0.5-1 cm in size. After this time, animals were injectedwith test article (0.1 ml, 20 MBq/ml or 200 MBq/ml) as an intravenousbolus via the tail vein. At various times p.i. animals were euthanased.Muscle, kidneys, urine, lung, liver, stomach, small intestine, largeintestine, thyroid and tumour were dissected and a blood sample taken.Dissected tissues, blood samples and standards were weighed and counted(Wallac Wizard). At least three animals per test point were studied.Results are expressed as % ID/g of tissue. Due to the fast clearance of^(99m)Tc-labelled CA1-ST₅₋₁₈ (greater than 90% excreted via urine 60mins p.i.) tumour uptake is also expressed as RR (FIG. 7).

Example 21 Imaging in Liver Metastases Model

Tumours were grown in animal as described in Example 20. Animals weremonitored for tumour growth for 8 to 10 weeks, until tumours were 0.5-1cm in size. After this time, animals were injected with test article(0.1 ml, 200 MBq/ml) as an intravenous bolus via the tail vein. Atvarious times p.i. animals were euthanased and whole body planar imagesacquired between 5 minutes and 24 hours p.i. using a gamma camera (ParkMedical Isocam-1). Image acquisition times were typically 15-30 min or150-250K counts (whole body, LEUHR collimator). The uptake of^(99m)Tc-labelled CA1-ST₅₋₁₈ in the rat liver metastases model is shown(FIG. 8). The image has the kidneys masked to improve the resolution ofthe imaged metastases.

Example 22 Uptake of CA1-ST₅₋₁₈ into a Non Colonic Tumour

To examine the specificity of the compounds of the present invention forcolonic carcinomas, C57BL/6 mice bearing sub-cutaneous Lewis lungtumours were injected with CA1-ST₅₋₁₈ and dissected at various timesp.i. Lewis lung is carcinoma that originally spontaneously arose in thelung of C57BL/6 mice and therefore is not of colorectal origin and isnot thought to express the ST receptor.

Example 23 ST Receptor Mediated Uptake of CAT-ST₅₋₁₈

To further examine the specificity of the compounds of the presentinvention for 5 colonic carcinomas, nude mice bearing T84 tumours asdescribed in Example 17 were imaged. Co-injection of 100 μg/mouse of STpeptide injected with CA1-ST₅₋₁₈ reduced the uptake by 40%. (FIG. 9).

Example 24 Effect of Impurities

It was observed that there was an increase in background tissue activityobserved with non-HPLC purified preparations; this could be potentiallydue to several factors: presence of excess peptide, presence of ^(99m)Tclabelled impurities (e.g. RHT or others). Further studies were carriedout to investigate the other possible causes for the increasedbackground seen with non-HPLC purified preparations, differentapproaches to reduce impurities such us RHT were tested. Preparationswere either filtered (0.2 μm filter size) or had MDP added, known toreduce RHT levels.

Example 25 Comparison of Tumour Model used with Human Tissue

T84 receptor density data show good comparison with those of human livermetastases (FIG. 10). Direct comparison of human liver metastases andxenograft tumour receptor density (Bmax) data clearly show that meanreceptor density of human tumours lies in between that seen for T84 andLS180 tumours.

In order to determine whether uptake varied with differences in receptordensity, biodistribution studies were undertaken in a range of xenografttumours with different ST receptor expressions.

1) A peptide-chelate conjugate comprising a peptide having affinity forthe ST receptor conjugated to a tetradentate chelating agent, whereinthe peptide having affinity for the ST receptor comprises 10 to 25 aminoacids. 2) The peptide-chelate conjugate of claim 1 wherein the peptidehaving affinity for the ST receptor comprises 13 to 19 amino acids. 3)The peptide-chelate conjugate of claim 1 where the tetradentatechelating agent is chosen from diaminedioximes, N₃S ligands, N₄ ligands,diaminediphenols and N₂S₂ ligands. 4) The peptide-chelate conjugate ofclaim 1 where the tetradentate chelating agent is chosen fromdiaminedioximes and N₃S ligands. 5) The peptide-chelate conjugate ofclaim 1 where the tetradentate chelating agent comprises ligands whichdeprotonate at basic pH to allow binding of ^(99m)Tc. 6) Thepeptide-chelate conjugate of claim 1 where the tetradentate chelatingagent is of formula A:

where R¹-R⁶ are all CH₃ and Q is —(CH₂)₂NR(CH₂)₂— and wherein R is an Rgroup as defined for Formula (A) in claim
 1. 7) The peptide-chelateconjugate of claim 1 where the tetradentate chelating agent is offormula B:

where R¹ and R² are both H and X is a thiol protecting group. 8) Thepeptide-chelate conjugate of claim 1 where the peptide is chosen fromSEQ ID Nos. 1 to 6 and FF—(CH₂)₅-SEQ ID No.
 7. 9) The peptide-chelateconjugate of claim 1 where the conjugation is either at the N-terminusor at the C-terminus of the peptide. 10) The peptide-chelate conjugateof claim 1 which further comprises a linker group between the peptideand the tetradentate chelating agent. 11) The peptide-chelate conjugateof claim 10 where the linker group comprises a peptide sequence of 5 to9 amino acids. 12) The peptide-chelate conjugate of claim 10 where thelinker group is selected from -poly-Lys-, -poly-Glu-,-(Gly)₂-Glu-(Lys)₃-, (Gly)₂-Glu-Lys-Glu-Lys-, (Phe)₂-(CH₂)₅-,(Lys)₆-Gly-, -(Gly)₃-(DGlu)₃- and -(Gly)₃-(aminocaproic acid)₂-. 13) Ametal complex which comprises a radiometal complexed to the tetradentatechelating agent of the peptide-chelate conjugate of claim
 1. 14) Themetal complex of claim 13 where the radiometal is chosen from ⁶⁴Cu, ⁶⁵Cuand ^(99m)Tc. 15) The metal complex of claim 13 where the radiometal is^(99m)Tc. 16) A radiopharmaceutical composition in a form suitable forhuman administration, which comprises the metal complex of claim
 13. 17)The radiopharmaceutical composition of claim 16, where the radiometal is^(99m)Tc. 18) Use of the radiopharmaceutical composition of claim 16 forimaging cancer of colorectal origin. 19) A kit for the preparation ofthe radiopharmaceutical composition of claim 16 which comprises: i) thepeptide-chelate conjugate of claim 1 ii) a reducing agent. 20) The kitof claim 19 where the reducing agent is a stannous salt. 21) The kit ofclaim 19 further comprising one or more of stabilisers; antioxidants;bulking agents for lyophilisation;